Characterization of the Nonendocrine Cell Populations in Human Embryonic Stem Cell–Derived (hESC) Islet-Like Clusters Posttransplantation

Abstract

Islet-like clusters derived from human embryonic stem cells (hESC) hold the potential to cure type 1 diabetes mellitus. Differentiation protocols of islet-like clusters lead to the generation of minor fractions of nonendocrine cells, which are mainly from endodermal and mesodermal lineages, and the risk of implanting these is unclear. In the present study, the histogenesis and the tumorigenicity of nonendocrine cells were investigated in vivo. Immunodeficient mice were implanted under the kidney capsule with islet-like clusters which were derived from differentiation of cells batches with either an intermediate or poor cell purity and followed for 8 or 26 weeks. Using immunohistochemistry and other techniques, it was found that the intermediate differentiated cell implants had limited numbers of small duct-like cysts and nonpancreatic tissue resembling gastrointestinal and retinal pigmented epithelium. In contrast, highly proliferative cystic teratomas were found at a high incidence at the implant site after 8 weeks, only in the animals implanted with the poorly differentiated cells. These findings indicate that the risk for teratoma formation and the amount of nonpancreatic tissue can be minimized by careful in-process characterization of the cells and thus highlights the importance of high purity at transplantation and a thorough ex-vivo characterization during cell product development.

Characterization of stem-cell-derived islets during differentiation and after implantation

“…Based on flow cytometry and scRNA-seq, the PPB was 60.3% ChgA + , 57.7% Nkx6.1 + , and C-peptide + with 5.1% Ki-67 + cells and 14.13% of cells co-expressing POU5F1, Nanog, and Sox2. the IPB was 97.5% ChgA + , 65.7% Nkx6.1 + , and C-peptide + with 0.2% Ki-67 + cells with no co-expression of POU5F1, Nanog, or Sox2 (Jensen et al, 2021).…”

“…To investigate the origin of the CA2 + CFTR À cells, Cdx2 and Sox2 were used as markers to distinguish the gastrointestinal tract phenotype and respiratory tract or cranial gastrointestinal tract; that is, the esophagus and stomach, respectively. The ductal epithelium in IPB-derived islet implants contained a few Cdx2 + cells, whereas PPB-derived islet implants contained whole segments positive for Cdx2 (Jensen et al, 2021). Similarly, IPB-derived islet implants showed no positive staining for Sox2, whereas PPB-derived islet implants showed segments of Sox2 + cells.…”

“…Despite the fact that 15%-50% of mice transplanted with SC-islets generate teratomas (Rezania et al, 2012;Kelly et al, 2011;Kroon et al, 2008), little is known about the histogenesis and function of non-endocrine cells. For this reason, Jensen et al (2021) differentiated, characterized, and transplanted two SC-islet batches with an intermediate or poor cell purity into mice to study the non-endocrine cells that arise upon transplantation. The poor-purity batch (PPB) and intermediate-purity batch (IPB) followed the same differentiation protocol except for cryopreservation and reaggregation, which were used for endocrine enrichment to generate the intermediate-purity SC-islets (Australia Intellectual Property Office Patent AU2018330499A1).…”

“…Review ll OPEN ACCESS Because of the endoderm induction and differentiation toward PPs, duct-like cysts were assumed to be of pancreatic origin (Pepper et al, 2019), which was evaluated through expression of human pancreatic duct and gallbladder markers, including cystic fibrosis transmembrane conductance regulator (CFTR) and carbonic anhydrase 2 (CA2), which is secreted by duct epithelia, in IPB and PPB islet transplants (Jensen et al, 2021). CFTR was expressed regionally in duct-like cysts, suggesting a regional pancreatic duct or gallbladder epithelium identity.…”

Mouse models and human islet transplantation sites for intravital imaging

“…Several additional models with various degrees of immune depletion have also been used for human islet transplant studies. This includes CB17.Cg- Prkdc scid Lyst bg-J /Crl (SCID beige) mice, a congenic line that results in defective NK cells, T cells, and B cells through mutations in Pkrdc and Lysbtg ( 79 , 83 , 84 ). Another immunocompromised mouse model occasionally used in human islet transplants is the CAnN.Cg- Foxn1 nu /Crl (BALB/c nu/nu) mouse model ( 85 , 86 ).…”